Abstract

The inactivation of microorganisms with treatments of temperature, pH and wateractivity is of vital importance to the food industry. In a previous study in fermentedmeat products (Ross et al., 2004), it was observed that temperature is the dominantfactor governing the rate of inactivation of E. coli. This study investigates whetherthis observation is true for other microorganisms and the mechanisms behind thephenomena. To better characterize and understand non-thermal inactivation, thisdissertation involves three main phases: kinetics investigation between two species,E. coli and L. monocytogenes, methods development (luminometry) and studies onmechanisms of non-thermal inactivation of L. monocytogenes, includingquantitative real time PCR (QPCR) and microarrays analysis.To study non-thermal inactivation kinetics, Listeria monocytogenes, a foodbornebacterial pathogen with different characteristics to E. coli, was selected in thisdissertation. Specifically, sixty-three inactivation rates were determined for bothspecies at a non-growth-permissive pH and water activity (pH 3.50 and aw 0.90respectively) at nine growth permissive temperatures. The results showed thatinactivation rates of both species were very similar, and the inactivation rateresponses of both were comparable to those previously and independently reportedfor a variety of E. coli strains under a wide range of growth-preventing pH andwater activity conditions (McQuestin, 2006). Thus, it appears that the influence ofnon-lethal temperature on the rate of inactivation of vegetative bacteria in inimicalenvironments is not species-dependent.For methodology, luminometry, in which intracellular ATP level is measured by itsability to generate light using the luciferin/luciferase enzyme system has beeninvestigated as means of quantifying microbial loads. To assess luminometry as amore rapid method of enumeration of bacteria in inimical environments,exponential phase L. monocytogenes ScottA and Fw 03/0035 were inactivatedunder inimical conditions (pH 3.50 and aw 0.90) at 25°C, 35°C and 45°C. Sampleswere periodically withdrawn for parallel viable count and luminometric analysis.The results showed that inactivation rates and kinetics determined by the ATPmethod were not comparable to those from viable counts. However, when bothmethods were applied to conditions permitting cell growth, there was a goodcorrelation. Thus, the A TP method is not sensitive enough to quantify microbialinactivation, particularly when cells are inactivated in sub-lethal conditions; but it iswell correlated with microbial growth.To better understand the physiology of bacterial cells in inimical environments,particularly whether they are metabolically active, tuf gene expression was studiedusing QPCR methods. L. monocytogenes strains ScottA and Fw 03/0035 wereinactivated with the same conditions as that described for the luminometryexperiments. Although viable cells numbers decreased from 108 to less than thedetection level (1.3 x 10 1 cells ml1), the tuf gene mRNA level remained unchanged.To determine whether this relatively high level of mRNA was due to unexpectedstability of the mRNA or due to de novo synthesis, additional experiments wereundertaken. Cells were inactivated under either mildly lethal temperature (55°C), inthe presence of rifampin (which inhibits DNA-dependent RNA polymerase) or a combination of both. The results show that when the antibiotic was present tuf geneexpression was reduced much more completely, with a three log reductioncompared with mildly lethal temperature with higher tuf gene levels of only a halflog reduction. This raises the possibility that L. monocytogenes under mildly lethalconditions of pH and aw or high temperature retain viability after being renderednon-culturable.To explore the genetic responses to the inactivation phenomena observed, genomicmicroarray analysis was performed to determine the effects oflow pH (3.5) and lowaw (0.90) on exponential phase L. monocytogenes ScottA, in a time-courseexperiment (5 min, 24 h, 48 h, 72 h).The results suggest that a large number ofgenes relevant to amino acid biosynthesis and metabolism are up-regulated,indicating a possible switch to alternative carbon sources as an energy supply andaid to maintenance of cell integrity. Genes belonging to the categories of structureand function of cell wall, cell movements, and carbohydrate metabolism were downregulated indicating lowered mobility. The regulatory network might play animportant role in regulating cellular physiological status and may dictate the rate ofinactivation.In this dissertation, the hypothesis that temperature is the main factor governing therate of inactivation of vegetative bacteria was firstly investigated and suggested tobe non-species dependent and this will be very useful to understand microbiologicalsafety of non-thermal processed food. For physiology of non-thermal inactivation,the thesis is fully addressed that when cells encounter environmental stresses, the regulatory network might play an important role in up-regulating and down-regulatinghouse keeping genes to cope with sublethal conditions and that whenthey reach the point of completely losing their culturability, they may still remainviable, thus entering the state of viable, but non-culturable, cells.

Item Type:

Thesis
(PhD)

Keywords:

Listeria monocytogenes, Food, Foodborne diseases

Copyright Holders:

The Author

Copyright Information:

Copyright 2008 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).

Additional Information:

Available for library use only and copying in accordance with the Copyright Act 1968, as amended. Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references. Ch. 1. Literature review -- Ch. 2. An investigation of the role of temperature in the inactivation rate of vegetative bacteria -- Ch. 3. Testing intracelullar ATP level as a rapid method for assessing microbial inactivation -- Ch. 4. Elongation factor EF-TU as an indicator of cell viability -- Ch. 5. Mechanisms of non-thermal inactivation - a time course study -- General summary and conclusion